1. Field of the Invention
This invention relates to a method of manufacturing a probe carrier by applying a probe solution to a specific position on a carrier, utilizing one or more than one indexes and an ink-jet method in particular. It also relates to a probe carrier manufactured by such a manufacturing method and a method of identifying the position of the target substance bonded to the probe on such a probe carrier by utilizing such indexes.
2. Related Background Art
When analyzing the base sequence of a gene DNA or conducting a gene diagnosis for a number of items simultaneously, probes of different types are needed to single out a DNA having a target base sequence in order to raise the reliability of operation. DNA microchips have been attracting attention as means for providing probes of a number of different types to be used for such sorting operations. A large number of solution specimens (e.g., 96, 384 or 1,536 specimens) containing proteins or drugs to be sorted normally have to be subjected to a screening operation in an orderly manner in the field of high throughput screening of chemicals or combinatorial chemistry. For these purposes, techniques of sequentially arranging a large number of different types of drugs, automatic screening technologies and dedicated devices for sorting the drugs arranged in this way and software for controlling a number of screening operations and statistically processing the obtained results have been and being developed.
Basically, such screening operations as described above that are conducted in parallel simultaneously consist in detecting an action or non-action or a response or non-response of each specimen to the known probes arranged in array, or probe array, provided as means for sorting the substances of specimens for evaluation under same conditions. Generally, the action or response to be used with each probe is defined in advance and therefore substances (drugs) of a same type are normally used as probe species that are mounted on a probe array. Then, the probe array may be that of DNA probes carrying a group of DNAs having different respective base sequences. DNAs, proteins and synthesized chemicals are examples of substances that may be used for a group of probes. While a probe array of a group of a plurality of probe species is used in many instances, a large number of identical DNAs having a same base sequence, identical proteins having a same amino acid sequence or identical chemical substances may be arranged in array depending on the type of screening operation. Such probes are mainly used for screening drugs.
In a probe array formed by a group of a plurality of probe species, a plurality of species of a group of DNAs having different base sequences, a group of proteins having different amino acid sequences, a group of different chemical substances or the like are often arranged in array on a substrate according to a predetermined order of arrangement. Particularly, DNA probe arrays are used for analyzing the base sequence of a gene DNA or conducting a gene diagnosis by analyzing a number of items simultaneously in order to raise the reliability of operation as pointed out above.
One of the problems to be solved for probe arrays formed by a group of a plurality of probe species is how to mount as many probes of different species, DNA probes having different base sequences for example, as possible on a single carrier. Differently stated, it is a problem of how to mount probes in array as densely as possible.
U.S. Pat. No. 5,424,186 describes a technique of preparing an array of DNA probes having respective base sequences that are different from each other by means of a stepwise elongation reaction of DNAs conducted on a carrier by utilizing photodecomposable protective groups and photolithography. With the proposed technique, it is possible to prepare a DNA probe array carrying DNAs of more than 10,000 different kinds that are different from each other in terms of base sequence per 1 cm2. The process of synthesizing a DNA by means of a stepwise elongation reaction, using this technique, comprises a photolithography step in which dedicated photomasks are used respectively for the four different kinds of base (A, T, C, G) in order to selectively elongate any of the bases at a predetermined position of the array so that consequently DNAs of different species having desired respective base sequences are synthetically produced and arranged on a substrate in a predetermined order. Then, the cost and the time required for preparing such a probe array rise as the DNA chain length increases. Furthermore, since the efficiency of nucleotide synthesis is not 100%, the ratio of DNAs that are defective in terms of the designed base sequence is not negligible. Additionally, when photodecomposable protective groups are used for the synthesis process, the efficiency of synthesis is rather poor if compared with the use of ordinary acid-decomposable protective groups. Therefore, the ratio of the DNAs that show the designed respective base sequences in the ultimately obtained array can be relatively small.
Besides, with the above identified known technique, since the products formed synthetically and directly on a carrier have to be used without any modification, it is not possible to sort out the DNAs having a defective base sequence from the DNAs having the designed respective base sequences and eliminate the former for the purpose of refining. There is also a problem that it is not possible to confirm the base sequences of the DNAs synthetically formed on the carrier and ultimately obtained as an array. This means that, if a base has not been subjected to predetermined elongation in a given elongation step probably because of an error or another in the step and hence the obtained probe array is not good, any screening operation using such a defective probe array gives rise to false results but there is no way of preventing such a problem from taking place. In short, absence of confirmation of base sequences is the largest and most intrinsic problem of the above identified known technique.
Apart from the above technique, techniques of manufacturing a probe array by synthesizing DNAs for probes in advance in a refined manner, confirming, if necessary, their respective base lengths and applying the DNAs to a carrier by means of an appropriate device such as a microdispenser are also known. PCT Patent Publication WO95/35505 describes a technique of applying DNAs onto a membrane by means of capillaries. With this technique, it is theoretically possible to prepare a DNA array having about 1,000 DNAs per 1 cm2. It is basically a technique of preparing a probe array by applying a probe solution to a predetermined position of a carrier for each probe by means of a single capillary-shaped dispensing device and repeating this operation. While no problem may arise when each probe is applied with a dedicated capillary-shaped dispensing device, a mutual contamination problem will occur if a small number of capillary-shaped dispensing devices are used repeatedly for the operation, so that the capillary-shaped dispensing devices have to be cleaned sufficiently each time a new probe species is brought in to avoid such a mutual contamination problem. Additionally, the position where each probe solution is applied needs to be controlled accurately. Therefore, this technique is not suited for preparing a probe array comprising a wide variety of probes that are arranged densely. Still additionally, the operation of applying a probe solution to the carrier is conducted by tapping the capillary tip to the carrier and hence not satisfactory in terms of both reproducibility and reliability.
There are also known techniques of applying a solution of a substance necessary for conducting an operation of DNA solid phase synthesis on a substrate in each elongation step by utilizing an ink-jet method. For example, European Patent Publication EP 0 703 825 B1 describes a technique of synthesizing DNAs of a plurality of different species having respective predetermined base sequences in a solid phase by applying nucleotide monomers and activators by means of respective piezo jet nozzles for the purpose of solid phase synthesis of DNAs. This application technique utilizing an ink-jet method is reliable in terms of reproducibility of the rate of application if compared with a solution application technique utilizing capillaries and also provides advantages for realizing high density probe arrays because the nozzle structure of the ink-jet system can be miniaturized. However, this technique is basically an application of a stepwise elongation reaction on a carrier and hence is not free from certain problems including that of being unable to confirm the base sequences of the DNAs synthetically formed on the carrier as pointed out earlier to be the largest problem or the technique according to U. S. Pat. No. 5,424,186. While the problem of conducting a cumbersome photolithography operation, using a dedicated mask in each elongation step, is dissolved with this technique, this technique is still accompanied to a certain extent by problems in terms of fixing predefined probes at respective positions, which is the requirement to be indispensably met for forming a probe array. It should be noted here that the above cited EP 0 703 825 B1 only describes a method of using a plurality of piezoelectric jet nozzles that are formed independently. The use of a small number of such nozzles is not suited for preparing high density probe arrays like the above described method of using capillary-shaped dispensing devices.
Japanese Patent Application Laid-open No. 11-187900 discloses a method of forming spots containing probes on a solid phase by causing droplets of probe-containing liquid to adhere to the solid phase by means of thermal ink-jet heads.
When preparing a probe array by utilizing an ink-jet method, it is desired from the viewpoint of high density arrangement to fix probes of as many different types as possible within a given area in order to improve the detection efficiency of diagnostic operations and avoid the need of preparing specimens to a large quantity. Additionally, it is necessary to accurately apply specific probes to respective intended positions from the viewpoint of reliability. It is also desired that only a right probe is fixed at an intended position from the viewpoint of eliminating diagnosis errors.
As pointed out above, more accurate position control will be required in the near future for fixing probes highly densely. However, with known probe fixing methods that utilize a conventional ink-jet process, it is often impossible to apply liquid accurately to a desired position as the liquid ejecting operation is conducted by regulating the relative positions of the carrier and the ink-jet head, visually confirming the posture of the entire carrier.
Additionally, when manufacturing a probe carrier by ejecting liquid in a number of times, using an ink-jet method, the carrier may have to be aligned with the ink-jet head for each liquid-ejecting operation. Then, when a liquid-ejecting operation is concluded, the entire probe formed by applying the liquid is positionally checked before the next liquid-ejecting operation is conducted. However, if the probe solution applied to the carrier by the last liquid-ejecting operation dries, it will no longer be possible to visually ascertain that the probe solution has been applied to the right position on the carrier. Therefore, the next liquid-ejecting operation has to be conducted before the probe solution applied to the carrier in the last liquid-ejecting operation dries and after visually confirming that the probe solution has been applied to the right position to make the manufacture of such a probe carrier disadvantageous. Furthermore, this technique is accompanied by additional problems including that the operation of visually aligning the carrier and the ink-jet head is time consuming and the number of points to be used for observing the alignment of the carrier and the ink-jet head is limited to make the alignment inaccurate. Moreover, if the probe carrier is turned upside down relative to the ink-jet head during the operation of manufacturing the probe carrier, it cannot be checked, if the carrier is transparent, simply by observing the carrier.
Japanese Patent Application No. 11-099000 discloses a method of fixing probes at intended respective positions by forming a division wall (also referred to as “black matrix”), or a light-shielding layer, on the carrier typically by means of photolithography in order to enhance the contrast produced by fluorescent light in the detection step. However, as a result of intensive research efforts, the inventors of the present invention found that, unless the ink-jet head is not accurately aligned relative to the probe carrier, the probe solutions of adjacently located apertures (wells) with a division wall interposed therebetween can become mixed with each other (to produce a mixed solution) so that it may no longer be possible to apply the proper probes to the intended position on the probe carrier. Then, the probe carrier will not function properly. Additionally, if the positions of ejection points are displaced, the probe solution in each well can be spread unevenly to expose the surface of the probe carrier over a large area. Then, there arise problems such as unreliable detection, difficulty of quantification, appearance of bright blank areas (also referred to simply as “blank areas”) and an increase of unspecified bonds. “Blank areas” are produced when the ejected probe solutions do not satisfactorily wet the probe carrier nor spread in the respective regions enclosed by a division wall. Then, probes will not be formed uniformly on the probe carrier as a result of applying probe solutions to the latter. When probes that are not uniformly formed are subjected to hybridization with respective specimens such as DNAs, the latter may not only become mated with the respective probes having a specific base sequence but also adhere to the substrate (e.g., glass) that is exposed in the blank areas to consequently reduce the contrast produced by fluorescent light during the operation of observation. FIGS. 3A and 3B of the accompanying drawings schematically illustrate this problem. In FIGS. 3A and 3B, reference symbols 31, 32, 33 and 34 respectively denote a transparent substrate, a division wall, a probe solution and a blank area. FIG. 3B is a cross sectional view taken along line 3B—3B in FIG. 3A.
Referring to FIGS. 3A and 3B, a blank area appears when a probe solution 33 does not wet corners showing a complex profile and/or a narrowly opened region nor spread well or when the probe solution 33 is applied thinly near the surrounding division wall 32. While the phenomenon of appearance of blank areas may be reduced when probe solutions are applied at an enhanced rate, it will not be totally eliminated.
Photoresist is typically used for forming a division wall 32. Therefore, various ingredients of photoresist can adhere to and remain in the inside of the openings of the division wall 32. The adherent residues can prevent probe solutions from wetting the probe carrier and spreading. Therefore, improvements are needed to obtain highly reliable fine probe carriers that can be manufactured with a high yield.
Furthermore, it is a problem to be solved urgently that the time required for aligning the ink-jet head and the probe carrier needs to be reduced because the operation of adjusting their relative positions and the use of a detector for detecting the overall profile of the black matrix for the purpose of alignment are time consuming and hence raise the manufacturing cost per chip.